Inflammation and Vascular Calcification Causing Effects of Oxidized HDL are Attenuated by Adiponectin in Human Vascular Smooth Muscle Cells

The role of oxidized high- density lipoprotein (oxHDL) and the protective effects of adiponectin in terms of vascular calcification is not well-established. This study was conducted to investigate the effects of oxHDL with regard to inflammation and vascular calcification and to determine the protective role of adiponectin in attenuating the detrimental effects of oxHDL. Cell viability, mineralization, and calcification assays were conducted to optimize the concentration of oxHDL. Then, human vascular smooth muscle cells (HAoVSMCs) were incubated with β-glycerophosphate, HDL, oxHDL, adiponectin, or the combination of oxHDL with adiponectin for 24 h. Protein expression of IL-6, TNF-α, osterix, RUNX2, ALP, type 1 collagen, osteopontin, osteocalcin, WNT-5a, NF-ĸβ(p65), cAMP and STAT-3 were measured by ELISA kits. OxHDL induced vascular calcification by promoting the formation of mineralization nodules and calcium deposits in HAoVSMCs. This was accompanied by an increased secretion of IL-6, osterix, WNT-5a and NF-ĸβ (p65). Interestingly, these detrimental effects of oxHDL were suppressed by adiponectin. Besides, incubation of adiponectin alone on HAoVSMCs showed a reduction of inflammatory cytokines, osteoblastic markers (RUNX2, osterix and osteopontin), WNT-5a and NF-ĸβ (p65). This study exhibits the ability of oxHDL in inducing inflammation and vascular calcification and these detrimental effects of oxHDL can be attenuated by adiponectin.

important event occurring during the advanced atherosclerosis stage (1). This plaque formation will lead to the formation of thrombotic occlusion which will narrow the blood vessel. The plaque would become unstable and rupture may occur; the blood flow to the brain or heart can be blocked; causing stroke or an infarct (2). Vascular calcification is a major risk factor for coronary artery disease and a major cause of death in patients with chronic kidney failure (3). It is believed to be initiated by transdifferentiation of vascular smooth muscle cells (VSMCs) to osteoblast-like cells (4) followed by calcium-hydroxyapatite crystal deposition in the medial layer of arteries. VSMCs, the predominant resident cells, play an important role in maintaining the blood flow and regulating contraction and relaxation. However, it has been shown that VSMCs isolated from atherosclerotic regions lost their physiological characteristics and so leading to stiffness of the arteries and reduced blood flow (5).
These isolated cells started to behave like osteoblast cells by secreting bone-related protein biomarkers including transcription factors for osteoblast differentiation and maturation (4)(5)(6). Increased reactive oxygen species, myeloperoxidase activity and pro-inflammatory cytokines such as IL-1, IL-6, TNF- and minimally oxidized LDL are some of the factors/substances so far identified for inducing vascular calcification (7,8).
HDL exerts many beneficial effects against the progression of cardiovascular disease by inhibiting the initial step of atherosclerosis progression through preventing accumulation of cholesterol in the macrophages via its reverse cholesterol efflux activity (8). While in the late stages of atherosclerosis, HDL plays its role in the prevention of vascular calcification by inhibiting the transdifferentiation of VSMCs (6). Furthermore, HDL also could reduce the alkaline phosphatase (ALP) secretion and signal transducer and activator of transcription 3 (STAT-3) activity in calcifying VSMCs isolated from atherosclerotic mice (7). However, HDL is prone to modification especially oxidation, which might change its protective properties into pro-atherogenic ones. The presence of oxidized high-density lipoprotein (oxHDL) in atherosclerotic plaques has been suspected as one of the major factors that enhanced the progression of atherosclerosis (1), even though little actual reports have been published. However, there is a study reporting that the presence of oxHDL heightened the activity of ALP and calcium deposition in already calcifying murine vascular cells (1).
Furthermore, increased HDL concentration in coronary artery disease patients by drugs such as fibrates and niacin failed to demonstrate its beneficial effects in reducing the risk of cardiovascular events. It is suggested that even though the quantity of HDL increases, the molecular structure of HDL is disturbed causing it to lose its beneficial components and become malfunctioning (9).
Adiponectin, an anti-inflammatory and antiatherogenic adipokine, has been known to exert many beneficial effects against the progression of atherosclerosis and its level in the circulation is inversely correlated with the progression of cardiovascular disease (10). Adiponectin reduced calcium deposition induced by TNF- and inorganic phosphate which significantly repressed the progression of vascular calcification (11  The mineralization assay was performed according to previous study (12). Briefly, the  While for the transcription factor, NF-ĸβ (p65), nuclear extracts were used and extracted by nuclear extraction kit (Cayman Chemical, USA).

Statistical analysis
Data were analysed using SPSS version 21.0.
ANOVA test was performed to assess overall differences between the different groups of treatment followed by Bonferroni Post-HOC test.
Significant value was set at P <0.05.

Non-toxic concentrations of oxHDL and adiponectin
The MTS test was performed to determine the non-toxic concentration range of oxHDL and adiponectin for further analysis. Figure  This phosphorous also induces the ALP activity, core binding factor α 1 (Cbfα1) gene expression, mineral nodules formation and calcium deposition in VSMCs, which mimic in vitro mineralization by osteoblasts (14).
In addition, incubation with 100 μg/ml protein oxHDL showed a significant calcium deposition in comparison with unstimulated group (P <0.0001) which was concurrent with the mineralization assay ( Figure 3). Therefore, the concentration of 100 μg/ml oxHDL was selected for further studies measuring the protein expression of proinflammatory and osteogenic biomarkers.

Effects of oxHDL and adiponectin on the secretion of the pro-inflammatory cytokines IL-6 and TNF-a
Elevation of pro-inflammatory cytokine secretion is known as one of the factors that heightens the progression of atherosclerosis. In this study, oxHDL significantly increased the secretion In contrast to the effect on IL-6 expression, oxHDL showed a non-significant increase of TNF-    osteocalcin. About 3x10 5 cells/well were treated for 24 h with β-glycerophosphate (positive control), media (un-stimulated, negative control), 100 μg/ml HDL, 100 μg/ml oxHDL, 5, 10 and 15 μg/ml adiponectin, and combination of 100 μg/ml oxHDL with 5, 10 and 15 μg/ml adiponectin. The cytosolic protein was extracted using AllPrep® RNA/Protein kit (Qiagen) and was quantified by using specific ELISA kit. The results are shown as meanSEM of 3 experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and **** P≤ 0.0001. *indicates the comparison with oxHDL group, # indicates the comparison with unstimulated group. Inflammation, caused by oxHDL, in this study elevated the secretion of WNT-5a in HAoVSMCs. IL-6 was shown to initiate the secretion of WNT-5a in melanoma cells and human bone marrow stromal cells, respectively (21,22). High secretion of WNT-5a is one of the indicators of the severity of atherosclerotic progression as it was found to be higher in advanced atherosclerotic plaque regions (23). It was also reported that the serum of atherosclerotic patients usually contains higher WNT-5a protein than normal individuals (23).
Interestingly, WNT-5a is also involved in inducing vascular calcification of smooth muscle cells through the non-canonical WNT-5a/ROR2 signaling pathway which supports our findings (23,24). Furthermore, oxLDL which is a very well- OxHDL also exhibited a neutral effect on all targeted osteoblast-related proteins: osteopontin, osteocalcin, type 1 collagen, and ALP. This may be due to the fact that RUNX2 is not affected by the treatment with oxHDL at least in the present study.
In bone cells (osteoblast), RUNX2 is a potent transcription factor in promoting the secretion of bone-related proteins targeted in this study (5).
Overall, oxidation causes HDL to lose its